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ARS Home » Pacific West Area » Wapato, Washington » Temperate Tree Fruit and Vegetable Research » Research » Research Project #425150

Research Project: Potato Germplasm Improvement for Disease Resistance and Superior Nutritional Content

Location: Temperate Tree Fruit and Vegetable Research

2018 Annual Report


Objectives
Objective 1: Apply genetic analyses, metabolic engineering, and targeted metabolic profiling to elucidate genetic, molecular, and biochemical factors governing host disease resistance and accumulation of select phytonutrients and vitamins in potatoes. Sub-objective 1.A. Characterize molecular and biochemical factors that modulate phytonutrient content. Sub-objective 1.B. Characterize molecular and biochemical mechanisms involved in disease/pest resistance. Objective 2: Evaluate, breed, and release potato germplasm with increased amounts of phytonutrients, which are suitable for the processing and fresh potato market, as well as for niche markets. Objective 3: Identify and release germplasm or varieties with improved resistance to powdery scab, black dot, Columbia root-knot nematode, zebra chip, potato mop top virus, potato cyst nematode, and examine the role of micronutrients in host resistance to Verticillium wilt. Sub-objective 3.A: Nematodes: Focus on identifying and developing germplasm, including trap crops, that can provide superior control options for Columbia root-knot nematode or Potato Cyst Nematode. Sub-objective 3B: Soil borne pathogens: Develop superior germplasm or management options for soil borne pathogens including powdery scab, potato mop top virus, black dot and Verticillium wilt. Objective 4: Determine available host-plant resistance and epidemiological parameters, and develop diagnostic tests for emerging pests and pathogens of potato such as zebra chip.


Approach
Objective 1: We will utilize molecular physiology approaches, including measuring gene expression, enzyme activity and metabolite pools by hyphenated techniques. Structural genes and regulatory genes will be assessed using transient assays or stable transgenics. The phenylpropanoid pathway will be a focus. HQT expression will be reduced using RNAi. LCMS will be used to assess differences in phenylpropanoids between wild type and silenced lines and the expression of at least 10-20 phenylpropanoid genes measured by qPCR. Another gene targeted for silencing will be dihydroflavonol-4-reductase (DFR). LCMS and GCMS analysis will be used to examine how phenylpropanoid and primary metabolism is reprogrammed in plants with altered DFR metabolism. MYB transcription factors will be identified in silico based on phylogenetic and protein similarity with known transcription factors. Function will be assessed in transient and stable assays. Compounds that cause the hatching of potato cyst nematode eggs will be partially purified from root extracts using chromatographic methods. Objective 2: Tuberling populations will be assembled and grown two successive seasons in the Klamath Basin of Oregon in unreplicated plots. Promising material will be analyzed for carotenoids, anthocyanins, antioxidants, and a range of other metabolites to select clones with high phytonutrient content. Statistically the data will be analyzed as a mixed model with locations, clones and interaction as fixed effects and reps within locations as random effects. We will use molecular markers to characterize hybrids and assure that we intercross only duplex Zep1 hybrids. Objective 3: We will combine PVY extreme resistance and CRKN resistant germplasm. The genetic nature will be explored by determining segregation ratios in reciprocal crosses. Mitochondrial fingerprinting will be expulsed as a diagnostic genetic marker of the restored phenotype. Crosses will be made to select a less spiny version of Solanum sisymbriifolium for use as a PCN trap crop. A. rhizogenes will be used to attempt to make a version of the plant with greater root mass. Hatching assays will be used to screen for other plants that may be a superior PCN trap crop. Crosses will be made to generate potatoes with resistance to Black dot and Powdery scab and evaluated in field trials with a randomized complete block design with four replications and ten plants per replication. The crown and root will be scored for degree of galling and sclerotia. The effect of micronutrient supplements on Verticillium wilt resistance will be assessed in field and greenhouse trials. Macro and micronutrients will be applied in-furrow. Objective 4: Psyllids collected during the survey and additional insects collected in the Pacific Northwest will be subjected to high resolution melt (HRM) analysis of the cytochrome oxidase gene in order to differentiate genetic variants of the psyllid. Extracts will be tested by PCR methods reported in the literature at dilutions up to 1,000 to determine level of sensitivity and reliability of the various methods on different host plant tissues.


Progress Report
This is the final report for this project as it has reached its five-year maximum term. For more information, see the new report for the new project, 2090-21220-002-00D, “Testing of Advanced Potato Germplasm for Biotic and Abiotic Resistances, Yield and Profitability Components”. The goal of this project was to develop superior new cultivars and to discover new knowledge that facilitates cultivar development with a special emphasis on improved disease resistance and nutritional value. The discoveries made over the five years of this project contributed to the enhanced sustainability of the nation’s potato industry, which provides hundreds of thousands of jobs. Consumers benefited from this research because reducing losses to pathogens keeps potatoes affordable as well as gaining from the development of potatoes with even higher amounts of vitamins, minerals and phytonutrients. Consumer food preferences have rapidly evolved over the last five years, as the effect of diet on health has received extensive coverage in the media, partly due to the nation’s ongoing obesity epidemic. This project’s emphasis on nutrition and specialty potatoes provides growers with the option to produce types of potatoes that can appeal to those consumers demanding novel and nutritious foods and are less interested in traditional products. Crossing blocks were established each year to generate seed that would be tested in extensive field trials in Washington, Oregon, Idaho, and elsewhere in the U.S. Breeding lines that have been released as cultivars from crosses made by ARS scientists at Prosser, Washington, include Yukon Nugget, TerraRosa and Castle Russet with many lines currently in advanced trials. Project scientists found that immature potatoes have considerably higher amounts of many phytonutrients than at maturity. Despite costing more, baby potatoes are valued by shoppers due to their perceived superior flavor. The effect of development on metabolites and gene expression of numerous different phytonutrients, including polyphenols, carotenoids, anthocyanins, protein, glycoalkaloids and folate were evaluated. These immature potatoes were harvested 60-80 days after planting, when typically, about one ounce in size and called “baby potatoes,” “gourmet potatoes,” or “new potatoes.” Baby potatoes can extend the appeal of potatoes to consumers who are not traditional buyers of potato products, because they are a more exotic, gourmet item, contain high phytonutrients, can be visually striking, cook faster and are smaller and eaten with skin on which requires less prep time. The development of specialty potatoes with higher amounts of phytonutrients intended for the fresh market was also investigated. Collectively, baby potatoes, and other specialty potatoes provide options for the industry to supply diverse types of potatoes that can meet changing consumer preferences and provide choices for those not interested in French fries or potato chips. While screening genotypes for their potential to be used for baby potato production, anthocyanin content up to 15-19 milligrams per gram (mg/g) dry weight (DW) was observed, and oxygen radical absorbance capacity (ORAC) values of over 300 micromoles (µmol) Trolox equivalents/g DW, values that rival spinach and kale. In screening of primitive potato germplasm and wild species a Phureja potato RN27.01 was discovered that contained a remarkable 41 mg/g of phenylprppanoids (PPs) and also high amounts of carotenoids. This potato has numerous defects and so is not suitable for commercialization, but, can be used as a source of traits. These exceptionally high amounts of phytonutrients showed that the phytonutrient amounts found in modern cultivars are likely not yet near the possible maximal amounts, given the amounts detected in primitive germplasm. ARS scientists in Prosser, Washington, worked with colleagues at Washington State University (WSU) to show that high-PP potatoes promote gut health. A taste panel analysis with collaborators at WSU demonstrated that purple and dark yellow flesh potatoes were as palatable as white fleshed potatoes. Advanced breeding materials were analyzed for the contents, stabilities, and broad-sense heritabilities of potassium and phosphorus, and results suggested these minerals can be increased by breeding. It is not understood what controls the trait for many of the important traits in potato. In the absence of such fundamental knowledge, one can compensate by using a brute force approach of generating extensive crosses and extensive screening. Our team worked towards understanding what controls important traits and made strides towards understanding factors that control tuber PP metabolism. For example, identifying a ternary transcription factor complex that regulates several branches of the pathway and establishing that stAN1 not only regulates anthocyanin synthesis but other phenolics, including chlorogenic acid. We made the unexpected discovery that sucrose appears to have a significant role in regulating tuber PP metabolism, which creates new opportunities to understand how environment influences PPs. It was established that among the possible biosynthetic pathways, chlorogenic acid only has one major route to its synthesis and that is through hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (HQT), and explored how carbon flux can be rerouted to different branches of the PP pathway. This knowledge will be useful in developing new cultivars with desired PP profiles and will be extended in the new project. Plant defense turns out to be vastly more complicated than could have been imagined a few decades ago, with a vast number of genes, networks and signal transduction pathways involved. As the mechanisms that plants use to resist disease are increasingly discovered, more options will be available for cultivar development. Highly conserved plant defense mechanisms that are regulated by salicylic acid or lipids were characterized and new mechanisms that lead to resistance to viruses, bacteria, and fungi identified with collaborators at the University of Kentucky. Another focus was potato cyst nematode, a quarantine pest discovered in Idaho. ARS researchers in Prosser, Washington, worked on trap crops and hatching factors and discovered there are over 15 distinct hatching factors produced by potatoes. Researchers developed a Litchi tomato trap crop that has reduced thorns and showed the fruit contains high amounts of phytonutrients which could provide an additional incentive for its use as a trap crop. DNA markers were identified for resistance to Columbia Root-knot Nematode (CRKN), an economically important pest of potato. We identified Solanum hougasii with collaborators at Oregon State University (OSU) as a new source of resistance genes to CRKN. Additionally, the resistance gene to race 1 of CRKN was tracked in the wild potato species, Solanum bulbocastanum, Solanum fendleri, and S. hougasii. This provided a different line of evidence that a bulbocastanum-like genome is present in tetraploid and hexaploid wild species. We participated in the discovery that transformation of potato with an RNAi effective in blocking the effector 16A10 causes a statistically significant reduction in nematode reproduction in both races of CRKN and both pathotypes in collaboration with scientists at WSU. Furthermore, the resistance was effective against a diverse array of tropical Meloidogyne species. With university and ARS collaborators across the U.S., as well as with international collaborators, ARS researchers in Prosser, Washington, advanced overall understanding of the zebra chip disease complex and the potato psyllid insect vector. A novel molecular technique, high resolution melting analysis, was utilized to map populations of the potato psyllid by comparing the psyllid mitochondrial cytochrome oxidase I. This work identified two new populations of the potato psyllid in the U.S., and found that four potato psyllid populations existed in geographically separate, but overlapping regions. This work mapped the psyllid populations on potato and the natural weed host, Solanum dulcamara, over time across central and western U.S., and also in Mexico and Central America. The results prompted numerous studies on the potato psyllid and its relationship to the zebra chip pathogen,‘Candidatus Liberibacter solanacearum’ (Lso) by external researchers and paved the way for a significant advancement in the knowledge of the insect and its role in zebra chip epidemiology. Additional accomplishments include improving molecular techniques for testing potato psyllids for the zebra chip pathogen, developing a fast insect nucleic acid extraction procedure and developing a potato psyllid genotyping tool for laboratories limited on equipment and reagents. With collaborators, the levels of Lso pathogen in potato psyllids collected from the central region of the U.S. were monitored several years and provided growers with a valuable tool for understanding the psyllid populations in their regions. Through additional collaborations, microbial communities were identified within the potato psyllid. Additional pathogens were studied. Through collaboration, the Potato virus Y (PVY) pathogen was identified in potato for the first time in Tajikistan and West Java, Indonesia, and with OSU researchers, an RNA-seq study identified differentially expressed genes during the early stages of PVY infection. Furthermore, studying population dynamics of plant pathogens and their movement into new regions or crops has identified Beet Leafhopper Virescence Agent (BLVA) in solanaceous pepper plants and its beet leafhopper insect vector in central Mexico for the first time. Additionally, with collaborators, as another first, the Potato mop top virus was detected in potatoes in Southeast Idaho. This progress summary provides the foundation and rationale for our new project.


Accomplishments
1. Isoprenoid metabolism in potatoes. Although isoprenoids are the largest group of plant natural products and have diverse functions, including influencing disease resistance, yields and nutritional value, their roles in potatoes have not been well-characterized. ARS scientists in Prosser, Washington, successfully developed transgenic potatoes that have increased or decreased expression of early genes in isoprenoid metabolism. Transgenic plants with increased isoprenoid metabolism tended to have larger size and tubers, and distinct roles for gene homologues were identified. Besides further elucidating the role of potato isoprenoid genes, these transgenic plants will allow us to test the possible role of isoprenoids in resistance to potato pests and pathogens for which there are currently no good options.

2. Identification of new Candidatus Liberibacter (Ca. L.) solanacearum haplotype. Zebra chip is an economically devastating disease of potatoes in the United States, caused by Ca. L. solanacearum haplotypes A and B. ARS researchers in Prosser, Washington, identified a new haplotype of Ca. L. solanacearum, haplotype F, in a single potato tuber originating from the U.S., that induces tuber symptoms indistinguishable from the typical zebra chip disease symptoms. Based on analysis of three different genes, it is closely related to the five known Ca. L. solanacearum haplotypes found around the world. The identification of this new genotype of the zebra chip pathogen adds another layer of complexity to zebra chip disease in the U.S. Further studies to identify host range and the specific insect vector capable of transmitting the pathogen are needed.

3. New source of nematode resistance discovered. Fumigation is the primary method used to control Meloidogyne chitwoodi, a widespread nematode potato pest that can reduce yields and cause tubers to be rejected due to nematode induced blemishes. ARS scientists in Prosser, Washington, and scientists from Oregon State University discovered a new source of resistance to M. chitwoodi; i.e., wild potato species, Solanum hougassi. It was discovered to be resistant to all pathotypes of M. chitwoodi tested. This offers a new source of resistance that can be introgressed into new cultivars to provide resistance to the nematode and potentially reduce the economic and environmental costs of fumigation.

4. Different roles of Candidatus Liberibacter (Ca. L.) solanacearum haplotypes in zebra chip disease incidence and tuber symptom severity. The economically devastating zebra chip disease in the U.S. has been attributed to infection by two different haplotypes of the bacterium, Ca. L. solanacearum, but little is known about the difference in disease epidemiology between haplotypes A and B. ARS researchers in Prosser, Wshington, conducted a large field cage experiment to identify differences in zebra chip incidence and symptom severity induced by the two Ca. L. solanacearum haplotypes. Both haplotypes A and B induced severe zebra chip tuber symptoms, but haplotype B was generally associated with more severe symptoms than haplotype A. Haplotype B also induced higher incidence of zebra chip symptoms and caused a reduction in tuber numbers compared with haplotype A. These results emphasize the importance of knowing what haplotype of Ca. L. solanacearum is present in a commercial field, as well as designating which haplotype is used in future studies on zebra chip disease.


Review Publications
Garczynski, S.F., Martin, J.A., Griset, M.L., Willett, L.S., Cooper, W.R., Swisher, K.D., Unruh, T.R. 2017. CRISPR/Cas9 editing of the codling moth (Lepidoptera: Tortricidae) CpomOR1 gene affects egg production and viability. Journal of Economic Entomology. 110(4):1847-1855.
Sun, X., Du, M., Navarre, D.A., Zhu, M. 2018. Purple potato extract promotes intestinal epithelial differentiation and barrier function by activating AMP-activated protein kinase. Molecular Nutrition and Food Research. 62(4):1700536. https://doi.org/10.1002/mnfr.201700536.
Boydston, R.A., Navarre, D.A., Collins, H.P., Chaves-Cordoba, B. 2017. The effect of vine kill method on vine kill, tuber skinning injury, tuber yield and size distribution, and tuber nutrients and phytonutrients in two potato cultivars grown for early potato production. American Journal of Potato Research. 95:54-70. https://doi.org/10.1007/s12230-017-9614-0.